The principal aim of the proposed research is to elucidate the molecular basis of visual excitation in mammalian retinal rod cells. We want to define the molecular and conformational links between the cis- trans isomerization of rhodopsin in the disc membrane and the change in sodium permeability of the outer membrane. It is evident that detailed information concerning the structure, dynamics, and enzymatic activities of photoreceptor membranes are needed. We propose to carry out the following spectroscopic and biochemical studies in this regard: (1) Energy transfer will be used as a spectroscopic ruler to map the structure of disc membranes and to detect conformational changes in rhodopsin. (2) The accessibility and role of the carbohydrate moiety of rhodopsin will be determined by using lectins (such as concanavalin A) as probes and glycosidases as modifiers. (3) Rhodopsin will be incorporated into model membrane systems to determine whether it acts as a light-controlled ion gate. (4) The reassembly of rhodopsin from a completely delipidated, unfolded apoprotein will be investigated by optical techniques. (5) Peptide mapping studies will be performed to determine whether rhodopsin contains distinct hydrophobic and hydrophilic domains. (6) The outer membrane of the rod outer segment will be isolated by affinity chromatography, and its constituents will be characterized. A number of proteins from the disc membrane and the interdisc space will be purified. (7) The effect of the transmembrane voltage on the enzymatic activities of rod outer segment enzymes such as adenyl cyclase and rhodopsin kinase will be investigated to determine whether the disc membrane potential can serve as a coupling mechanism.